Monitoring Anti-PEG Antibodies Level upon Repeated Lipid Nanoparticle-Based COVID-19 Vaccine Administration.

PEGylated lipids are one of the four constituents of lipid nanoparticle mRNA COVID-19 vaccines. Therefore, various concerns have been raised on the generation of anti-PEG antibodies and their potential role in inducing hypersensitivity reactions following vaccination or in reducing vaccine efficacy due to anti-carrier immunity. Here, we assess the prevalence of anti-PEG antibodies, in a cohort of vaccinated individuals, and give an overview of their time evolution after repeated vaccine administrations. Results indicate that, in our cohort, the presence of PEG in the formulation did not influence the level of anti-Spike antibodies generated upon vaccination and was not related to any reported, serious adverse effects. The time-course analysis of anti-PEG IgG showed no significant booster effect after each dose, whereas for IgM a significant increase in antibody levels was detected after the first and third dose. Data suggest that the presence of PEG in the formulation does not affect safety or efficacy of lipid-nanoparticle-based COVID-19 vaccines.

Maraviroc as a potential HIV-1 latency-reversing agent in cell line models and ex vivo CD4 T cells.

Recent studies have suggested that the CCR5 antagonist maraviroc (MVC) may exert an HIV-1 latency reversal effect. This study aimed at defining MVC-mediated induction of HIV-1 in three cell line latency models and in ex vivo CD4 T cells from six patients with suppressed viraemia. HIV-1 induction was evaluated in TZM-bl cells by measuring HIV-1 LTR-driven luciferase expression, and in ACH-2 and U1 latently infected cell lines by measuring cell-free (CFR) and cell-associated (CAR) HIV-1 RNA by qPCR. NF-κB p65 was quantified in nuclear extracts by immunodetection. In ex vivo CD4 T cells, CAR, CFR and cell-associated DNA (CAD) were quantified at baseline and 1-7-14 days post-induction (T1, T7, T14). At T7 and T14, the infectivity of the CD4 T cells co-cultured with MOLT-4/CCR5 target cells was evaluated in the TZM-bl assay (TZA). Results were expressed as fold activation (FA) with respect to untreated cells. No LTR activation was observed in TZM-bl cells at any MVC concentration. NF-κB activation was only modestly upregulated (1.6±0.4) in TZM-bl cells with 5 µM MVC. Significant FA of HIV-1 expression was only detected at 80 µM MVC, namely on HIV-1 CFR in U1 (3.1±0.9; P=0.034) and ACH-2 cells (3.9±1.4; P=0.037). CFR was only weakly stimulated at 20 µM in ACH-2 (1.7±1.0 FA) cells and at 5 µM in U1 cells (1.9±0.5 FA). Although no consistent pattern of MVC-mediated activation was observed in ex vivo experiments, substantial FA values were detected sparsely on individual samples with different parameters. Notably, in one sample, MVC stimulated all parameters at T7 (2.3±0.2 CAD, 6.8±3.7 CAR, 18.7±16.7 CFR, 7.3±0.2 TZA). In conclusion, MVC variably induces HIV-1 production in some cell line models not previously used to test its latency reversal potential. In ex vivo CD4 T cells, MVC may exert patient-specific HIV-1 induction; however, clinically relevant patterns, if any, remain to be defined.

Computational Analysis of Multiparametric Flow Cytometric Data to Dissect B Cell Subsets in Vaccine Studies.

The generation of the B cell response upon vaccination is characterized by the induction of different functional and phenotypic subpopulations and is strongly dependent on the vaccine formulation, including the adjuvant used. Here, we have profiled the different B cell subsets elicited upon vaccination, using machine learning methods for interpreting high-dimensional flow cytometry data sets. The B cell response elicited by an adjuvanted vaccine formulation, compared to the antigen alone, was characterized using two automated methods based on clustering (FlowSOM) and dimensional reduction (t-SNE) approaches. The clustering method identified, based on multiple marker expression, different B cell populations, including plasmablasts, plasma cells, germinal center B cells and their subsets, while this profiling was more difficult with t-SNE analysis. When undefined phenotypes were detected, their characterization could be improved by integrating the t-SNE spatial visualization of cells with the FlowSOM clusters. The frequency of some cellular subsets, in particular plasma cells, was significantly higher in lymph nodes of mice primed with the adjuvanted formulation compared to antigen alone. Thanks to this automatic data analysis it was possible to identify, in an unbiased way, different B cell populations and also intermediate stages of cell differentiation elicited by immunization, thus providing a signature of B cell recall response that can be hardly obtained with the classical bidimensional gating analysis. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Genotyping of Toxoplasma gondii strain directly from human CSF samples of congenital toxoplasmosis clinical case.

This report describes a case of congenital toxoplasmosis in a newborn in Southern Italy. A pregnant mother had been admitted at the 20th week of her pregnancy on account of pharyngodynia and laterocervical lymphadenopathy. Although serological testing of the mother's serum documented a seroconversion with positive IgG and IgM anti-Toxoplasma antibodies during II trimester, the woman refused to perform prenatal diagnosis for congenital toxoplasmosis. Fetal ultrasound scan already showed mild asymmetrical triventricular hydrocephaly and cerebral calcifications. After birth, real-time PCR on cerebrospinal fluid and blood samples of the newborn showed a positive result for 529bp-repeat element DNA of T. gondii, In addition brain magnetic resonance imaging and computed tomography showed a characteristic diffuse brain tissue loss associated with hydrocephalus. For the first time molecular characterization of T. gondii isolate was performed directly from the newborn's CSF samples by using nested-PCR-RFLP of sag-2 and pk1 genes. The PCR-RLFP analysis revealed that the isolate belongs to the clonal type II, the predominant lineage causing human toxoplasmosis, as confirmed by DNA sequencing.

Fitness Cost of Rifampin Resistance in Neisseria meningitidis: In Vitro Study of Mechanisms Associated with rpoB H553Y Mutation.

Rifampin chemoprophylaxis against Neisseria meningitidis infections led to the onset of rifampin resistance in clinical isolates harboring point mutations in the rpoB gene, coding for the RNA polymerase ß chain. These resistant strains are rare in medical practice, suggesting their decreased fitness in the human host. In this study, we isolated rifampin-resistant rpoB mutants from hypervirulent serogroup C strain 93/4286 and analyzed their different properties, including the ability to grow/survive in different culture media and in differentiated THP-1 human monocytes and to compete with the wild-type strain in vitro. Our results demonstrate that different rpoB mutations (H553Y, H553R, and S549F) may have different effects, ranging from low- to high-cost effects, on bacterial fitness in vitro. Moreover, we found that the S549F mutation confers temperature sensitivity, possibly explaining why it is observed very rarely in clinical isolates. Comparative high-throughput RNA sequencing analysis of bacteria grown in chemically defined medium demonstrated that the low-cost H553Y substitution resulted in global transcriptional changes that functionally mimic the stringent response. Interestingly, many virulence-associated genes, including those coding for meningococcal type IV pili, porin A, adhesins/invasins, IgA protease, two-partner secretion system HrpA/HrpB, enzymes involved in resistance to oxidative injury, lipooligosaccharide sialylation, and capsular polysaccharide biosynthesis, were downregulated in the H553Y mutant compared to their level of expression in the wild-type strain. These data might account for the reduced capacity of this mutant to grow/survive in differentiated THP-1 cells and explain the rarity of H553Y mutants among clinical isolates.

Identification of Inquilinus limosus in cystic fibrosis: a first report in Italy.

Cystic fibrosis is a genetic disorder associated with a polymicrobial lung infection where classical pathogens and newly identified bacteria may interact. Inquilinus limosus is an a-proteobacterium recently isolated in the airways of cystic fibrosis patient. We report the first case in Italy of I.limosus isolation from the sputum sample of a cystic fibrosis patient. The patient is a 20-years-old man with cystic fibrosis, regularly attending the Regional Care Center for Cystic Fibrosis at the Federico II University Hospital of Naples. Microbiological culture methods detected a mu- coid gram negative bacillus in the patient's sputum sample. The isolate exhibited a distinct antimicrobial suscep- tibility profile with a high MIC for several drugs. The MALDI-TOF mass spectrometry analysis indicated the bac- terium isolated as I. limosus, confirmed by 16s rDNA sequence analysis. The described clinical case demonstrates how the bacterial biodiversity in the airways of cystic fibrosis patients is still underestimated. Cystic fibrosis lung represents an ecological niche suitable for growth of a wide variety of unusual bacteria not commonly associated with human diseases, such as I. limosus. Therefore further studies are needed to evaluate the epidemiology and clinical implications of I. limosus in the physiopathology of cystic fibrosis lung infection.

Spike-Specific Memory B Cell Response in Hematopoietic Cell Transplantation Recipients following Multiple mRNA-1273 Vaccinations: A Longitudinal Observational Study.

Preventing SARS-CoV-2 infection is of utmost importance in allogeneic hematopoietic cell transplantation patients (allo-HCT), given their heightened susceptibility to adverse outcomes associated with SARS-CoV-2 infection. However, limited data are available regarding the immune response to COVID-19 vaccines in these subjects, particularly concerning the generation and persistence of spike-specific memory response. Here, we analyzed the spike-specific memory B cells in a cohort of allo-HCT recipients vaccinated with multiple doses of the mRNA-1273 vaccine and monitored the spike-specific antibody response from baseline up to one month after the fourth dose. After the primary vaccine series, the frequency of spike-specific B cells, detected within the pool of Ig-switched CD19+ cells, significantly increased. The booster dose further induced a significant expansion, reaching up to 0.28% of spike-specific B cells. The kinetics of this expansion were slower in the allo-HCT recipients compared to healthy controls. Spike-specific IgG and ACE2/RBD binding inhibition activity were observed in 80% of the allo-HCT recipients after the first two doses, with a significant increase after the third and fourth booster doses, including in the subjects who did not respond to the primary vaccine series. Additionally, 87% of the allo-HCT recipients exhibited positive cross-inhibition activity against the BA.1 variant. Our findings provide evidence that allo-HCT recipients need repeated doses of the mRNA-1273 vaccine to induceSARS-CoV-2 specific immune response similar to that observed in healthy individuals. This is particularly crucial for vulnerable individuals who may exhibit a limited response to the primary series of SARS-CoV-2 vaccination.

Homologous or heterologous administration of mRNA or adenovirus-vectored vaccines show comparable immunogenicity and effectiveness against the SARS-CoV-2 Omicron variant.

BACKGROUND: Heterologous prime-boost schedules have been employed in SARS-CoV-2 vaccination, yet additional data on immunogenicity and effectiveness are still needed. RESEARCH DESIGN AND METHODS: Here, we measured the immunogenicity and effectiveness in the real-world setting of the mRNA booster dose in 181 subjects who had completed primary vaccination with ChAdOx1, BNT162b2, or mRNA1273 vaccines (IMMUNO_COV study; protocol code 18,869). The spike-specific antibody and B cell responses were analyzed up to 6 months after boosting. RESULTS: After an initial slower antibody response, the heterologous ChAdOx1/mRNA prime-boost formulation elicited spike-specific IgG titers comparable to homologous approaches, while spike-specific B cells showed a higher percentage of CD21-CD27- atypical cells compared to homologous mRNA vaccination. Mixed combinations of BNT162b2 and mRNA-1273 elicited an immune response comparable with homologous strategies. Non-significant differences in the Relative Risk of infection, calculated over a period of 18 months after boosting, were reported among homologous or heterologous vaccination groups, indicating a comparable relative vaccine effectiveness. CONCLUSIONS: Our data endorse the heterologous booster vaccination with mRNA as a valuable alternative to homologous schedules. This approach can serve as a solution in instances of formulation shortages and contribute to enhancing vaccine strategies for potential epidemics or pandemics.

Streptococcus pyogenes Φ1207.3 Is a Temperate Bacteriophage Carrying the Macrolide Resistance Gene Pair mef(A)-msr(D) and Capable of Lysogenizing Different Streptococci.

Streptococcus pyogenes prophage Φ1207.3 (formerly Tn1207.3) carries the mef(A)-msr(D) resistance genes, responsible for type M macrolide resistance. To investigate if Φ1207.3 is a functional bacteriophage, we transferred the element from the original S. pyogenes host in a prophage-free and competence-deficient S. pneumoniae strain. Pneumococcal cultures of the Φ1207.3-carrying lysogen were treated with mitomycin C to assess if Φ1207.3 enters the lytic cycle. Mitomycin C induced a limited phage burst and a growth impairment, resulting in early entrance into the stationary phase. To determine if Φ1207.3 is able to produce mature phage particles, we prepared concentrated supernatants recovered from a mitomycin C-induced pneumococcal culture by sequential centrifugation and ultracentrifugation steps. Negative-staining transmission electron microscopy (TEM) of supernatants revealed the presence of phage particles with an icosahedral, electron-dense capsid and a long, noncontractile tail, typical of a siphovirus. Quantification of Φ1207.3 was performed by quantitative PCR (qPCR) and semiquantitatively by TEM. PCR quantified 3.34 × 104 and 6.06 × 104 excised forms of phage genome per milliliter of supernatant obtained from the untreated and mitomycin C-treated cultures, respectively. By TEM, we estimated 3.02 × 103 and 7.68 × 103 phage particles per milliliter of supernatant. The phage preparations of Φ1207.3 infected and lysogenized pneumococcal recipient strains at a frequency of 7.5 × 10-6 lysogens/recipient but did not show sufficient lytic activity to form plaques. Phage lysogenization efficiently occurred after 30 min of contact of the phages with the recipient cells and required a minimum of 103 phage particles. IMPORTANCE Bacteriophages play an important role in bacterial physiology and genome evolution. The widespread use of genome sequencing revealed that bacterial genomes can contain several different integrated temperate bacteriophages, which can constitute up to 20% of the genome. Most of these bacteriophages are only predicted in silico and are never shown to be functional. In fact, it is often difficult to induce the lytic cycle of temperate bacteriophages. In this work, we show that Φ1207.3, a peculiar bacteriophage originally from Streptococcus pyogenes, which can lysogenize different streptococci and carries the macrolide resistance mef(A)-msr(D) gene pair, is capable of producing mature virions, but only at a low level, while not being able to produce plaques. This temperate phage is probably a partially functional phage, which seems to have lost lytic characteristics to specialize in lysogenization. While we are not used to conceiving phages separately from lysis, this behavior could actually be more frequent than expected.

Does Nirmatrelvir/Ritonavir Influence the Immune Response against SARS-CoV-2, Independently from Rebound?

Recurrence of coronavirus disease 19 (COVID-19) symptoms and SARS-CoV-2 viral load relapse have been reported in people treated with nirmatrelvir/ritonavir (NM/r). However, little is understood about the etiology of this phenomenon. Our aim was to investigate the relation between the host's immune response and viral rebound. We described three cases of COVID-19 rebound that occurred after treatment with nirmatrelvir/ritonavir (group A). In addition, we compared spike-specific antibody response and plasma cytokine/chemokine patterns of the rebound cases with those of (i) control patients treated with nirmatrelvir/ritonavir who did not show rebound (group B), and (ii) subjects not treated with any anti-SARS-CoV-2 drug (group C). The anti-spike antibodies and plasma cytokines/chemokines were similar in groups A and B. However, we observed a higher anti-BA.2 spike IgG response in patients without antiviral treatment (group C) [geometric mean titer 210,807, 5.1- and 8.2-fold higher compared to group A (p = 0.039) and group B (p = 0.032)]. Moreover, the patients receiving antiviral treatment (groups A-B) showed higher circulating levels of platelet-derived growth factor subunit B (PDGF-BB) and vascular endothelial growth Factors (VEGF) and lower levels of interleukin-9 (IL-9), interleukine-1 receptor antagonist (IL-1 RA), and regulated upon activation normal T cell expressed and presumably secreted chemokine (RANTES) when compared to group C. In conclusion, we observed lower anti-spike IgG levels and different cytokine patterns in nirmatrelvir/ritonavir-treated patients compared to those not treated with anti-SARS-CoV-2 drugs. This suggests that early antiviral treatment, by reducing viral load and antigen presentation, could mitigate the immune response against SARS-CoV-2. The clinical relevance of such observation should be further investigated in larger populations.

B cell response after SARS-CoV-2 mRNA vaccination in people living with HIV.

BACKGROUND: Limited longitudinal data are available on immune response to mRNA SARS-CoV-2 vaccination in people living with HIV (PLWHIV); therefore, new evidence on induction and persistence of spike-specific antibodies and B cells is needed. METHODS: In this pilot study we investigated the spike-specific humoral and B cell responses up to six months after vaccination with two doses of mRNA vaccines in 84 PLWHIV under antiretroviral therapy compared to 79 healthy controls (HCs). RESULTS: Spike-specific IgG persisted six months in PLWHIV with no significant differences compared to HCs, even though a significantly lower IgG response was observed in patients with CD4+ T cells < 350/mmc. The frequency of subjects with antibodies capable of inhibiting ACE2/RBD binding was comparable between PLWHIV and HCs a month after the second vaccine dose, then a higher drop was observed in PLWHIV. A comparable percentage of spike-specific memory B cells was observed at month six in PLWHIV and HCs. However, PLWHIV showed a higher frequency of spike-specific IgD- CD27- double-negative memory B cells and a significantly lower rate of IgD- CD27+ Ig-switched memory B cells compared to HCs, suggesting a reduced functionality of the antigen-specific memory B population. CONCLUSIONS: The mRNA vaccination against SARS-CoV-2 elicits humoral and B cell responses quantitatively similar between PLWHIV and HCs, but there are important differences in terms of antibody functionality and phenotypes of memory B cells, reinforcing the notion that tailored vaccination policies should be considered for these patients.

SARS-CoV-2 vaccination has been demonstrated to protect people from severe COVID-19 and death. This is achieved through the induction of a specific immune response that recognizes and responds to the virus. Limited data are available on the immune response to SARS-CoV-2 vaccination in people living with HIV (PLWHIV). In this study, we evaluated the immune response up to six months after vaccination with two doses of vaccines in PLWHIV being treated with the standard antiretroviral therapy. We show that the immune response observed in PLWHIV is broadly similar to that in healthy subjects but that there are some differences in the cells induced as part of the immune response. We therefore suggest that specific vaccination policies should be considered for these PLWHIV.

Trajectory of Spike-Specific B Cells Elicited by Two Doses of BNT162b2 mRNA Vaccine.

The mRNA vaccines for SARS-CoV-2 have demonstrated efficacy and immunogenicity in the real-world setting. However, most of the research on vaccine immunogenicity has been centered on characterizing the antibody response, with limited exploration into the persistence of spike-specific memory B cells. Here we monitored the durability of the memory B cell response up to 9 months post-vaccination, and characterized the trajectory of spike-specific B cell phenotypes in healthy individuals who received two doses of the BNT162b2 vaccine. To profile the spike-specific B cell response, we applied the tSNE and Cytotree automated approaches. Spike-specific IgA+ and IgG+ plasmablasts and IgA+ activated cells were observed 7 days after the second dose and disappeared 3 months later, while subsets of spike-specific IgG+ resting memory B cells became predominant 9 months after vaccination, and they were capable of differentiating into spike-specific IgG secreting cells when restimulated in vitro. Other subsets of spike-specific B cells, such as IgM+ or unswitched IgM+IgD+ or IgG+ double negative/atypical cells, were also elicited by the BNT162b2 vaccine and persisted up to month 9. The analysis of circulating spike-specific IgG, IgA, and IgM was in line with the plasmablasts observed. The longitudinal analysis of the antigen-specific B cell response elicited by mRNA-based vaccines provides valuable insights into our understanding of the immunogenicity of this novel vaccine platform destined for future widespread use, and it can help in guiding future decisions and vaccination schedules.

The third dose of mRNA SARS-CoV-2 vaccines enhances the spike-specific antibody and memory B cell response in myelofibrosis patients.

Vaccination against SARS-CoV-2 using mRNA-based vaccines has been highly recommended for fragile subjects, including myelofibrosis patients (MF). Available data on the immune responsiveness of MF patients to mRNA SARS-CoV-2 vaccination, and the impact of the therapy with the JAK inhibitor ruxolitinib, are still fragmented. Here, we profile the spike-specific IgG and memory B-cell response in MF patients, treated or not with ruxolitinib, after the second and the third dose of SARS-CoV-2 BNT162b2 (BioNTech) and mRNA-1273 (Moderna) vaccines. Plasma and peripheral blood mononuclear cells samples were collected before vaccination, post the second and the third doses and tested for spike-specific antibodies, ACE2/RBD antibody inhibition binding activity and spike-specific B cells. The third vaccine dose significantly increased the spike-specific IgG titers in both ruxolitinib-treated and untreated patients, and strongly enhanced the percentage of subjects with antibodies capable of in vitro blocking ACE2/RBD interaction, from 50% up to 80%. While a very low frequency of spike-specific B cells was measured in blood 7 days after the second vaccination dose, a strong and significant increase was elicited by the third dose administration, generating a B cell response similar to the one detected in healthy controls. Despite the overall positive impact of the third dose in MF patients, two patients that were under active concomitant immunosuppressive treatment at the time of vaccination, and a patient that received lymphodepleting therapies in the past, remained low responders. The third mRNA vaccine dose strongly increases the SARS-CoV-2 specific humoral and B cell responses in MF patients, promoting a reactivation of the immune response similar to the one observed in healthy controls.

Short or Long Interval between Priming and Boosting: Does It Impact on the Vaccine Immunogenicity?

Characterizing the impact of the vaccination schedule on the induction of B and T cell immune responses is critical for improving vaccine immunogenicity. Here we compare the effect of a short (4 weeks) or a long (18 weeks) interval between priming and boosting in mice, using a model vaccine formulation based on the chimeric tuberculosis vaccine antigen H56 combined with alum. While no significant difference was observed in serum antigen-specific IgG response and the induction of antigen-specific T follicular helper cells into draining lymph nodes after the two immunization schedules, a longer interval between priming and boosting elicited a higher number of germinal center-B cells and H56-specific antibody-secreting cells and modulated the effector function of reactivated CD4+ T cells. These data show that the scheduling of the booster immunization could affect the immune response elicited by vaccination modulating and improving the immunogenicity of the vaccine.

Evidence of SARS-CoV-2-Specific Memory B Cells Six Months After Vaccination With the BNT162b2 mRNA Vaccine.

SARS-CoV-2 mRNA vaccines have demonstrated high efficacy and immunogenicity, but limited information is currently available on memory B cell generation and long-term persistence. Here, we investigated spike-specific memory B cells and humoral responses in 145 subjects, up to 6 months after the BNT162b2 vaccine (Comirnaty) administration. Spike-specific antibodies peaked 7 days after the second dose and significant antibody titers and ACE2/RBD binding inhibiting activity were still observed after 6 months, despite a progressive decline over time. Concomitant to antibody reduction, spike-specific memory B cells, mostly IgG class-switched, increased in the blood of vaccinees and persisted 6 months after vaccination. Following the in vitro restimulation, circulating memory B cells reactivated and produced spike-specific antibodies. A high frequency of spike-specific IgG+ plasmablasts, identified by computational analysis 7 days after boost, positively correlated with the generation of IgG+ memory B cells at 6 months. These data demonstrate that mRNA BNT162b2 vaccine elicits strong B cell immunity with spike-specific memory B cells that still persist 6 months after vaccination, playing a crucial role for a rapid response to SARS-CoV-2 virus encounter.

The Slower Antibody Response in Myelofibrosis Patients after Two Doses of mRNA SARS-CoV-2 Vaccine Calls for a Third Dose.

Immunization with mRNA SARS-CoV-2 vaccines has been highly recommended and prioritized in fragile subjects, including patients with myelofibrosis (MF). Available data on the vaccine immune response developed by MF patients and the impact of ruxolitinib treatment are still too fragmented to support an informed decision on a third dose for this category of subjects. Here, we show that 76% of MF patients develop spike-specific IgG after the second mRNA SARS-CoV-2 vaccine dose, but the response has a slower kinetics compared to healthy subjects, suggesting a reduced capability of their immune system to promptly react to vaccination. A reduced ACE2/RBD binding inhibition activity of spike-specific antibodies was also observed, especially in ruxolitinib-treated patients. Our results, showing slow kinetics of antibody responses in MF patients following vaccination with mRNA SARS-CoV-2 vaccines, support the need for a third vaccine dose.

Optimized Protocol for the Detection of Multifunctional Epitope-Specific CD4+ T Cells Combining MHC-II Tetramer and Intracellular Cytokine Staining Technologies.

Analysis of multifunctional CD4+ T cells is fundamental for characterizing the immune responses to vaccination or infection. Major histocompatibility complex (MHC)/peptide tetramers represent a powerful technology for the detection of antigen-specific T cells by specific binding to their T-cell receptor, and their combination with functional assays is fundamental for characterizing the antigen-specific immune response. Here we optimized a protocol for the detection of multiple intracellular cytokines within epitope-specific CD4+ T cells identified by the MHC class II tetramer technology. The optimal procedure for assessing the functional activity of tetramer-binding CD4+ T cells was based on the simultaneous intracellular staining with both MHC tetramers and cytokine-specific antibodies upon in vitro restimulation of cells with the vaccine antigen. The protocol was selected among procedures that differently combine the steps of cellular restimulation and tetramer staining with intracellular cytokine labeling. This method can be applied to better understand the complex functional profile of CD4+ T-cell responses upon vaccination or infection.

Type M Resistance to Macrolides Is Due to a Two-Gene Efflux Transport System of the ATP-Binding Cassette (ABC) Superfamily.

The mef(A) gene was originally identified as the resistance determinant responsible for type M resistance to macrolides, a phenotype frequently found in clinical isolates of Streptococcus pneumoniae and Streptococcus pyogenes. MefA was defined as a secondary transporter of the major facilitator superfamily driven by proton-motive force. However, when characterizing the mef(A)-carrying elements Tn1207.1 and Φ1207.3, another macrolide resistance gene, msr(D), was found adjacent to mef(A). To define the respective contribution of mef(A) and msr(D) to macrolide resistance, three isogenic deletion mutants were constructed by transformation of a S. pneumoniae strain carrying Φ1207.3: (i) Δmef(A)-Δmsr(D); (ii) Δmef(A)-msr(D); and (iii) mef(A)-Δmsr(D). Susceptibility testing of mutants clearly showed that msr(D) is required for macrolide resistance, while deletion of mef(A) produced only a twofold reduction in the minimal inhibitory concentration (MIC) for erythromycin. The contribution of msr(D) to macrolide resistance was also studied in S. pyogenes, which is the original host of Φ1207.3. Two isogenic strains of S. pyogenes were constructed: (i) FR156, carrying Φ1207.3, and (ii) FR155, carrying Φ1207.3/Δmsr(D). FR155 was susceptible to erythromycin, whereas FR156 was resistant, with an MIC value of 8 µg/ml. Complementation experiments showed that reintroduction of the msr(D) gene could restore macrolide resistance in Δmsr(D) mutants. Radiolabeled erythromycin was retained by strains lacking msr(D), while msr(D)-carrying strains showed erythromycin efflux. Deletion of mef(A) did not affect erythromycin efflux. This data suggest that type M resistance to macrolides in streptococci is due to an efflux transport system of the ATP-binding cassette (ABC) superfamily, in which mef(A) encodes the transmembrane channel, and msr(D) the two ATP-binding domains.

Heterologous Prime-Boost Combinations Highlight the Crucial Role of Adjuvant in Priming the Immune System.

The induction and modulation of the immune response to vaccination can be rationally designed by combining different vaccine formulations for priming and boosting. Here, we investigated the impact of heterologous prime-boost approaches on the vaccine-specific cellular and humoral responses specific for a mycobacterial vaccine antigen. C57BL/6 mice were primed with the chimeric vaccine antigen H56 administered alone or with the CAF01 adjuvant, and boosted with H56 alone, or combined with CAF01 or with the squalene-based oil-in-water emulsion adjuvant (o/w squalene). A strong secondary H56-specific CD4+ T cell response was recalled by all the booster vaccine formulations when mice had been primed with H56 and CAF01, but not with H56 alone. The polyfunctional nature of T helper cells was analyzed and visualized with the multidimensional flow cytometry FlowSOM software, implemented as a package of the R environment. A similar cytokine profile was detected in groups primed with H56 + CAF01 and boosted with or without adjuvant, except for some clusters of cells expressing high level of IL-17 together with TNF-α, IL-2, and IFN-γ, that were significantly upregulated only in groups boosted with the adjuvants. On the contrary, the comparison between groups primed with or without the adjuvant showed a completely different clusterization of cells, strengthening the impact of the formulation used for primary immunization on the profiling of responding cells. The presence of the CAF01 adjuvant in the priming formulation deeply affected also the secondary humoral response, especially in groups boosted with H56 alone or o/w squalene. In conclusion, the presence of CAF01 adjuvant in the primary immunization is crucial for promoting primary T and B cell responses that can be efficiently reactivated by booster immunization also performed with antigen alone.